CN107192822A - Detection of Salmonella proficiency testing sample and preparation method thereof in medicine - Google Patents
Detection of Salmonella proficiency testing sample and preparation method thereof in medicine Download PDFInfo
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- CN107192822A CN107192822A CN201710372893.6A CN201710372893A CN107192822A CN 107192822 A CN107192822 A CN 107192822A CN 201710372893 A CN201710372893 A CN 201710372893A CN 107192822 A CN107192822 A CN 107192822A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
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Abstract
Detection of Salmonella proficiency testing sample and preparation method thereof in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine.Detection of Salmonella proficiency testing sample includes object bacteria and background flora in medicine, and the object bacteria is Salmonella enteritidis, and the background flora is made up of EHEC, Klebsiella Pneumoniae, staphylococcus aureus, Bacillus cereus.Uniformity of the present invention, stability meet proficiency testing requirement, can at utmost ensure the stability of test sample；The preparation method of sample, technique is simple, and success rate is high.
Description
Technical field
Detection of Salmonella ability in field of quality control in terms of the invention belongs to microbiologic inhibition tests, more particularly to a kind of medicine
Verification sample and preparation method thereof.
Background technology
Medicine microbiological Test field is very special.At present, although have accreditation body both at home and abroad and organize the micro- life of medicine
Thing proficiency testing and the large-scale precedent for preparing microorganism proficiency testing sample, but sample is the pure bacterium only containing detection of Salmonella
Strain, exists without background bacterium, is differed greatly with actual sample, i.e. detection of Salmonella standard sample still belongs to blank field.
Detection of Salmonella, belongs to enterobacteriaceae, gram-negative enteric bacillus.If in the detection of Salmonella sample prepared, target flora
Easily change (breeding and death etc.), and the storage life that may result in sample is short, and the uniformity and stability of sample are poor.Therefore,
Consider the biochemical characteristic of bacterium, choose the metastable flora of property as target flora to ensure sample uniformity and
Stability is particularly significant.The process specifications of detection of Salmonella sample preparation are higher, and the uniformity and stability of sample with
Lyophilized strain, lyophilized process conditions, the use of freeze drying protectant, medium of rehydration etc. have close relationship.
Detection of Salmonella index in medicine, directly reflects the hygienic quality and potential pathogenic risk of medicine.If husky in medicine
Door bacterium number can cause patient's direct infection detection of Salmonella, or even have fatal risk more than a threshold quantity.Therefore, uniformity is prepared
The detection of Salmonella sample all good with stability, the random and uncertainty that detection of Salmonella can be avoided to examine, truly reflection detection
The ability in laboratory.This to improve Good Laboratory controlled level, ensure drug safety, even break International trade practices,
Improving China's medicine international competitiveness all has special important meaning.
The content of the invention
The purpose of the present invention is the number of bacteria for overcoming work total in detection of Salmonella determination sample in transport, storage and test etc.
During clump count there is provided detection of Salmonella proficiency testing sample, uniformity, stability in a kind of medicine the problem of can all change
Meet proficiency testing requirement, it is a further object to provide the preparation method of the sample, technique is simple, and success rate is high.
The technical scheme that is used to achieve the above object of the present invention is：Detection of Salmonella proficiency testing sample in medicine, it is special
Levying is：Including object bacteria and background flora, the object bacteria is Bacterium enteritidis (Salmonella enteritidis), institute
Background flora is stated by EHEC (E.coil), Klebsiella Pneumoniae (Klebsiella pnenmoniae), golden yellow grape
Coccus (Staphylococcus aureus), Bacillus cereus (Bacillus cereus) composition.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein trehalose volume fraction is 12%, degreasing
Milk powder volume fraction is 0.5%.
The aimed concn of object bacteria is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
The preparation method of detection of Salmonella proficiency testing sample in a kind of medicine, it is characterized in that：The choosing of bacterial strain is added including sample
Select, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, wherein sample was freezed
Cheng Wei：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, and detailed process is：
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to recovery
Bacterial strain is identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3CFU/mL prepares bacteria suspension, the bacteria suspension of object bacteria and the bacteria suspension of 4 background bacterium is prepared respectively, respectively
The bacteria suspension of background bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension is pressed with background flora suspension
1:1 volume ratio is mixed, and obtains plastc ring, is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle, adds bottle
Plug, but space is left, reality should not be covered；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
The freeze drying protectant is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Marine alga
Sugar 12%, skimmed milk power 0.5%.
The uniformity of the sample and stability test are according to CNAS-GL03《Proficiency testing sample homogeneity and stability
Evaluation guide》Carry out.
Strain of the present invention selection using Bacterium enteritidis as detection of Salmonella sample object bacteria, with EHEC,
Klebsiella Pneumoniae, staphylococcus aureus, Bacillus cereus are used as background flora.It is well known that Salmonella intestines bar
Cordycepps, gram-negative enteric bacillus.
The detection of Salmonella proficiency testing sample of the present invention has the advantage that characteristic：Microorganism living, quantity do not change,
Biochemical character does not morph, and sets about from the condition for meeting special transport, passes through the research of special process, stability and uniformity
Research etc. form detection of Salmonella (qualitative) item in the sample preparation technology of a set of perfect proficiency testing, entirely appropriate development medicine
Purpose is examined, correct evaluating ability the result.
Brief description of the drawings
Fig. 1 is present invention process flow chart.
Fig. 2 is the stability test result figure of sample detection of Salmonella under the conditions of 4 DEG C.
Fig. 3 is the stability test result figure of sample at different temperatures.
Embodiment
Below in conjunction with the accompanying drawings and specific embodiment is described in further detail to the present invention, but the invention is not limited in tool
Body embodiment.
Embodiment 1
Detection of Salmonella proficiency testing sample in medicine, including object bacteria and background flora, the object bacteria are Salmonella enteritidis
(Salmonella enteritidis), the background flora is by EHEC (E.coil), Klebsiella Pneumoniae
(Klebsiella pnenmoniae), staphylococcus aureus (Staphylococcus aureus), Bacillus cereus
(Bacillus cereus) is constituted.
The sample is using trehalose, skimmed milk power and sterilized water as matrix, and wherein trehalose volume fraction is 12%, degreasing
Milk powder volume fraction is 0.5%.
The aimed concn of object bacteria is 10 in the sample2CFU/mL, the aimed concn of background flora is 103CFU/mL。
Embodiment 2
As shown in figure 1, in a kind of embodiment 1 in medicine detection of Salmonella proficiency testing sample preparation method, including sample addition bacterium
The selection of strain, the preparation of freeze drying protectant, sample are lyophilized, four steps of the uniformity of sample and stability test, specific steps
It is as follows：
1st, sample adds the selection of bacterial strain
According to object bacteria：Salmonella enteritidis (Salmonella enteritidis), background flora：ETEC
(E.coil), Klebsiella Pneumoniae (Klebsiella pnenmoniae), staphylococcus aureus (Staphylococcus
Aureus), Bacillus cereus (Bacillus cereus) selection standard bacterial strain, all reference cultures are purchased from government and specified
Mechanism, and with bacterial strain certificate, it is ensured that the traceability of bacterial strain.
2nd, the preparation of freeze drying protectant
Sample is using trehalose, skimmed milk power and sterilized water as matrix (volume fraction：Trehalose 12%, skimmed milk power 0.5%) match somebody with somebody
Freeze drying protectant processed.
3rd, sample is freezed
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar, it is recovered and is grown at 36 ± 1 DEG C, and to the bacterial strain of recovery
Identified；
(2) bacterium is increased
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
(3) bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3CFU/mL prepares bacteria suspension.To ensure the bacterial strain containing above aimed concn in obtained final sample,
According to object bacteria 10 in the present embodiment2CFU/mL prepares the bacteria suspension of object bacteria；Background bacterium presses 103CFU/mL prepares 4 background bacterium
Bacteria suspension, by 1:1 volume ratio is mixed, and forms background flora suspension；Background flora suspension presses 1 with target bacteria suspension:1 volume ratio
Mixing, obtains plastc ring, and bacterial strain content is checked using turbidimetry；It is placed in be stirred continuously down on magnetic stirring apparatus and takes 1.0mL
It is dispensed into sample bottle (cillin bottle), adds bottle stopper, but to leave space, reality should not be covered；
(4) freeze and pack
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, and freeze drier parameter is by following setting：
Chilling rate (Cooling Rate) 0.5 DEG C/min
- 1 DEG C of 15min of early stage cold point (Incipient Freezing Point)
- 40 DEG C of cryogenic temperature (Cooling temperature)
- 29 DEG C of eutectic point (Melting Point Eutectic temperature)
20 DEG C of 120min of heating-up temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, whole freeze-drying process 40h.
After sample freeze-drying terminates, directly carry out jumping a queue in case；Closing machine.The not tight cillin bottle of plug is rejected,
Sample is vacuum state in cillin bottle；
(5) store
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, and the sample of preservation suitably manage and detect, from whole sample
Random selected sample carries out uniformity and stability test in product, and satisfaction requires that after discharge carries out laboratory monitoring to laboratory of participating in the experiment
Compare.
4th, the uniformity of sample and stability test
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.Check
Sample homogeneity and stability are according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability guide》Carry out.
Gained sample homogeneity and Detection of Stability method and result are as follows：
12 samples are randomly selected respectively, and using SN/T 1897-2007 methods, the sramana of 2 × 12 parts of samples is tested in repeat condition
Bacterium.Result data carries out statistical disposition with one-way analysis of variance (ANOV), and statistic procedure and result are as follows：
The variance analysis formula of table 1
In upper table,
The sample homogeneity testing result of table 2
The sample homogeneity of table 3 tests the results of analysis of variance
Conclusion：Under 95% fiducial probability, compared with influence of the other factors to test result, the inhomogeneities of sample is to connect
Receive.
Using two kinds of stability test：One kind is the stability test at short temperature (4 DEG C), another
Kind it is the stability test at high temperature (traffic condition of analog sample), from three temperature spots, respectively 20 DEG C,
36 DEG C and 45 DEG C.Periodic detection sample, tests 3 samples, by 2 × 3 parts of samples for the different temperature points different holding time
As a result average (logarithmic transformed after) and uniformity test results contrast, between absolute difference divided by test plan in ability comment
Valency is with sane standard deviation S*, it is that principle determines to meet proficiency testing sample requirement under condition of different temperatures that ratio, which is less than 0.3,
The most long holding time.Stability test result is shown in Fig. 2 and Fig. 3.
Embodiment 3
In medicine described in the present embodiment each step of the preparation method of detection of Salmonella proficiency testing sample with phase in embodiment 2
Together, different technical parameters is：Strain cultures select nutrient agar slant medium；During bacteria suspension is prepared, target bacteria suspension
The concentration of middle object bacteria is according to 5 × 102CFU/mL is prepared, and the concentration of each background bacterium is according to 10 in background flora suspension3 CFU/mL
Prepare；Freeze-drying process 45h.
Embodiment 4
In medicine described in the present embodiment each step of the preparation method of detection of Salmonella proficiency testing sample with phase in embodiment 3
Together, different technical parameters is：During bacteria suspension is prepared, the concentration of object bacteria is according to 4.5 × 10 in target bacteria suspension2CFU/mL matches somebody with somebody
System；Freeze-drying process 50h.
Claims (6)
1. detection of Salmonella proficiency testing sample in medicine, it is characterized in that：Including object bacteria and background flora, the object bacteria is enteritis
Salmonella, the background flora is by EHEC, Klebsiella Pneumoniae, staphylococcus aureus, Bacillus cereus group
Into.
2. detection of Salmonella proficiency testing sample in medicine according to claim 1, it is characterized in that：The sample with trehalose,
Skimmed milk power and sterilized water are matrix, and wherein trehalose volume fraction is that 12%, skimmed milk power volume fraction is 0.5%.
3. detection of Salmonella proficiency testing sample in medicine according to claim 1 or 2, it is characterized in that：Target in the sample
The aimed concn of bacterium is 102CFU/mL, the aimed concn of background flora is 103 CFU/mL。
4. the preparation method of detection of Salmonella proficiency testing sample in medicine according to claim 1, it is characterized in that：Including sample
Add that the selection of bacterial strain, the preparation of freeze drying protectant, sample be lyophilized, four steps of the uniformity of sample and stability test, its
Middle sample freeze-drying process is：Bacterial strain recovery passage-increasing bacterium-bacteria suspension prepares-lyophilized and packaging-storage, and detailed process is：
（1）Bacterial strain recovery passage
Reference culture is added and is inoculated into nutrient agar slant medium, it is recovered and is grown at 36 ± 1 DEG C, and to multiple
The bacterial strain of Soviet Union is identified；
（2）Increase bacterium
Object bacteria culture to the logarithmic growth end of term, background bacteria group culture to stationary phase scrapes bacterium colony from inclined-plane, is added to 10mL jelly
The bacterium solution of corresponding single culture is made in dry protective agent；
（3）Bacteria suspension is prepared
The bacterium solution that a upper process is obtained is mixed with freeze drying protectant, according to target the aimed concn 10 of bacterium2CFU/mL and background bacterium
The aimed concn 10 of group3 CFU/mL prepares bacteria suspension, the bacteria suspension of object bacteria and the bacteria suspension of 4 background bacterium is prepared respectively, respectively
The bacteria suspension of background bacterium presses 1:1 volume ratio is mixed to form background flora suspension, then target bacteria suspension is pressed with background flora suspension
1:1 volume ratio is mixed, and obtains plastc ring, is placed in be stirred continuously down on magnetic stirring apparatus and is dispensed into sample bottle, adds bottle
Plug, but space is left, reality should not be covered；
（4）Lyophilized and packaging
The sample bottle that will be equipped with plastc ring is uncapped and is put into freeze dryer, is freeze-dried 40~50h, is tied when sample is freeze-dried
Shu Hou, directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
（5）Storage
It is kept in dark place under the conditions of sample is placed on into -18 DEG C, random selected sample carries out uniformity and stably from whole samples
Property experiment, satisfaction require after discharge to participate in the experiment laboratory carry out contrasting between laboralories.
5. the preparation method of detection of Salmonella proficiency testing sample in medicine according to claim 4, it is characterized in that：It is described lyophilized
Protective agent is the sterilized water containing trehalose and skimmed milk power, and wherein volume fraction is respectively：Trehalose 12%, skimmed milk power
0.5%。
6. the preparation method of detection of Salmonella proficiency testing sample in medicine according to claim 4, it is characterized in that：The sample
Uniformity and stability test according to CNAS-GL03《Proficiency testing sample homogeneity and estimation of stability guide》Carry out.
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Cited By (1)
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| CN110596227A (en) * | 2019-08-08 | 2019-12-20 | 河北省食品检验研究院（国家果类及农副加工产品质量监督检验中心、河北省食品安全实验室） | Preparation method of time-of-flight mass spectrum ready-to-use type salmonella stanliensis qualitative standard sample |
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